Regenerating neruons in the leech can form functional connections with their normal targets, thus restoring at the level of single cells precisely the wiring established during development. Just what steps a neuron takes as it locates and synapses with its proper targets, what roles are played by surrounding glia, neighboring neurons, and target neurons, and how the new synapses compare with the old in structure, distribution and function are the questions the proposed project addresses. The methodology will include extracellular and intracellular electrophysiological recording and intracellular injection of markers for subsequent light- and electron microscopic examination. Regeneration will be studied in culture as well as within the leech; single cells can be killed with intracellular injection of proteases. Complex patterns of regenerated synaptic contact between sensory and motor cells will be reconstructed with a computer. Particularly well suited for tracing the regeneration of a specific synapse is the system of S-interneurons, which form electrical synapses with each other at precise locations within the c.n.s. and whose axons have a distinctive size, shape and position. An injured S-cell sometimes rapidly restores function by forming an electrical synapse on its severed axon. We plan to investigate under what conditions the nervous system can thus short-circuit the normal regeneration mechanism, whether neuroglial cells and other types of neurons can similarly regenerate, and whether the severed axon segment serves as a guide for regeneration. A related problem is how a severed axon segment survives, whether by glial nourishment, flow of substances from other neurons, or simply by low temperature at which regeneration occurs. An understanding of the mechanisms for accurate regeneration in the leech c.n.s. might suggest ways that neural regeneration in higher animals could be made reliable and precise.